The invasion of the human body by microbial substances and the resulting infection is often difficult to diagnose because of diverse or minimal pathological effects. Immunology, the study of immune responses to the introduction of foreign substances to the body, concerns itself with two classes of macromolecules. These classes, antigens and antibodies, react in specific manners. The foreign invading substance or antigen has generally two properties associated with it, immunogenicity, the capacity to stimulate the formation of a corresponding antibody, and the ability to react specifically with those antibodies. The antibody itself is a protein that is formed in response to the presence of an antigen for reaction with that specific antigen. Antibodies belong to a special group of serum proteins called immunoglobulins. Although the group of antibodies comprises a restricted group of proteins that are capable of specifically reacting with antigens, there is an enormous variety of macromolecules capable of behaving as antigens including proteins, many polysaccharides, nucleoproteins, lipoproteins, numerous synthetic polypeptides as well as many other small molecules when they are suitably linked to proteins or synthetic polypeptides.
The specificity of antibody-antigen reactions has been utilized in the diagnosis of pathological states or physiological conditions and more particularly, in the determination of the presence of antigenic determinants, i.e. those restricted portions of the antigen molecule that determine the specificity of antibody-antigen reactions. In accordance with the knowledge of those skilled in the art, antigen-antibody reactions can be manifested by enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunofluorescence (IF) techniques with high sensitivity, however, these techniques are complex and generally require great skill, time and complex electronic instruments.
Typically, the detection of a soluble antigen present in solution may be effected by the addition of an antibody specific for that antigen at sufficient concentrations to permit realization of the specific antigen-antibody reaction. The antigens and antibodies combine and form large insoluble aggregates which become visible as precipitates. Unfortunately, if the antigen is present only at low concentration levels, the resulting weak antigen-antibody reaction may not form a precipitate of sufficient size and quality to permit direct visible detection. In order to enhance the size of the precipitate and thereby aid in its detection, it has been found that the agglutination of antigen and an antibody linked with another particle such as an inert latex particle forms a significantly larger precipitate more clearly visible to the naked eye.
The agglutination immunoassay utilizing inert particles made from a latex or polystyrene polymer poses other problems. The polystyrene latex carrier particles are difficult to coat and standardize and additionally often give false results due to nonspecific reactions because of physical adherence by hydrophobic materials to the polystyrene latex carrier particles in aqueous media. Alternatively, erythrocytes, e.g. red blood cells, have been advantageously used as indicator particles since they are convenient passive carriers of antigens. There are numerous erythrocyte-linked antigen settling tests or passive hemagglutination assays which have succeeded primarily because the red blood cells can be preserved by chemical fixation and most antigens existing on the surface of the RBC can tolerate the chemical treatment without loosing their affinity to specific antibody. Additionally, antigens can often be chemically attached to red blood cells thus permitting a cross-linkage to an indicator particle subsequent to immunological reaction with the specific antibody.
These methods and principles have been described in publications such as "The Preparation and Use of Formalinized Erythrocytes with Attached Antigens or Haptens to Titrate Antibodies" by Joseph S. Ingraham, published in Proc. Soc. Exp. Biol. Med., Vol. 99, 1958, p. 452-456; "The Use of a Water-Soluble Carbodiimide as a Coupling Reagent in the Passive Hemagglutination Test" by H. M. Johnson et al., published in the Journal of Immunology, Vol. 97, No. 6, 1966, p. 791-796.
There has, however, been little success in chemically linking antibodies to particles such as red blood cells without damaging the avidity of the antibody. For instance, the reverse passive hemagglutination assay (RPHA) reagent with antibody against hepatitis-B surface antigens (HBsAg) on aldehyde-fixed red blood cells for the diagnosis of hepatitis-B is an example of a test which met the requirements for "third generation" sensitivity of hepatitis tests in the U.S., however, not only is the test less sensitive than existing RIA hepatitis tests, but the resulting cell settling patterns are extremely difficult to read by those inexperienced in the techniques. One of the reasons for this is thought due to an inability to covalently link antibody onto chemically activated particles or cells with either control over the sites or the orientation of the immunoglobulin molecules on the surface. Thus, the antibody molecules are likely to have random orientation as well as multiple attachments on the carrier surface. Hence, the avidity of the antibody and its ability to form a lattice structure in agglutination is seriously impaired.
It is one of the objects of the present invention to provide a multiple or conjugate antibody reagent having an antibody linked to a carrier particle primarily by single attachment, thereby providing flexible orientation and minimal effect on conformation and another antibody of differing specificity free for further immunological reactions.
Use of multiple antibodies in the past has been essentially limited to head-to-tail combinations as compared to the present invention which combines antibodies in a substantially tail-to-tail fashion. For instance, U.S. Pat. No. 4,048,298 to Niswender teaches a procedure directed toward a solid phase double antibody competitive radioimmunoassay procedure wherein the antigenic substance or ligand is converted to a radioactive derivative and used to stimulate the production of antibodies. Thereafter a second antibody specific for the first antibody is produced. The second antibody may be immobilized onto a water insoluble organic polymeric substance or one having a polymeric nature. The second antibody is capable of reacting with the first antibody-antigen complex and converting all attached substances into an insoluble form. It is believed the activity of the second antibody is probably diminished because attachment by covalent bonding or physical adsorption is nonspecific and occurs with multiple attachment sites, resulting in distortion of the conformation and flexibility of the antibody molecule. Additionally, the use of radioisotopes presents problems which are preferably avoided. Specifically, these problems include (a) the relatively short life of gamma emitting isotopes (e.g. .sup.125 I), (b) impairment of immunological reactivity and specificity by gamma radiation of the isotope, (c) health hazards involved in the use of dangerous radioisotopes which necessitates the use of procedures complying with federal standards as well as requiring precise safety controls and (d) expensive, complex instrumentation such as scintillation counters.
The present invention has, as one of its objectives, the reduction of the complexity of the reagent, the test procedure, and the preferable avoidance of a radioisotope in order to accomplish a similar sensitivity.
The prior art has attempted to increase sensitivity using radioisotopes as described above as well as fluorescent and enzyme indicators and hybrid antibodies.
Multivalent hybrid antibodies have been produced previously and typical technology is reported in Molecular Immunology, Vol. 17, 1980, p. 395-401, in an article entitled "Multivalent Hybrid Antibody" by Ghetie and Mota. The publication describes the formation of a multivalent hybrid antibody having dual specificity using protein A of Staphylococcus aureus (SpA). The hybrid is made by immunoglobulin (IgG) reaction with SpA which is capable of linking the IgG molecules through their Fc portions. For example, anti-A and anti-B antibodies were reacted with SpA and formed a multivalent hybrid antibody having a molecular composition described as (IgG anti-A/SpA/IgG anti-B).sub.2. This reagent is not, however, suitable for agglutination tests or attachment to an indicator particle because of nonspecific agglutination which can be caused by the multivalent antibody of the hybrid in the presence of a single type of antigen. The present invention teaches the use of a univalent antibody such as an Fab' fragment of IgG for attachment onto carrier particles in order to avoid nonspecific agglutination. This is not possible with the Ghetie and Mota system since reaction with SpA requires the presence of the Fc portion of the immunoglobulin which portion is not present in a univalent Fab' fragment.
Nisonoff and Rivers, in Archives of Biochemistry and Biophysics, Vol. 93, 1961, p. 460-462, cited by Ghetie and Mota, describe the preparation of a univalent hybrid antibody molecule having dual specificity by the recombination of two Fab' fragments from the reduction of anti-A (Fab').sub.2 and anti-B (Fab').sub.2. Problems associated with this approach include random combination as well as interference by unwanted anti-A/anti-A and anti-B/anti-B recombinants. An object of the present invention includes the avoidance of these problems by the use of a hetero-bifunctional coupling reagent capable of conjugating two different specific immunoglobulins. Hetero-bifunctional coupling reagents have been described in the following publications: J. Carlsson et al., Biochemistry Journal, Vol. 173, 1978, p. 723-737; G. Ishikawa et al., Enzyme Labeled Immunoassay of Hormones and Enzymes, ed. S. B. Pal, 1978, p. 43-57; T. Kitagawa and T. Aikawa, Journal of Biochemistry, Vol. 79, 1976, p. 233-235; J. N. Limet et al., Journal of Immunology Methods, Vol. 28, 1979, p. 25-32 ; E. S. Rector et al., Journal of Immunology Methods, Vol. 24, 1978, p. 321-336; and S. Yoshitake et al., European Journal of Biochemistry, Vol. 101, 1979, p. 395-399.
"Lectin Immuno Test" by J. L. Guesdon and S. Avramas published in the Journal of Immunological Methods, Vol 39, 1980, p. 1-13 describes yet another possible method of detecting antigens utilizing fresh red blood cells as a marker. The publication discloses the use of a concanavalin A antibody conjugate which utilizes the combining sites present on lectin to bind the marker substance. The proposed method and reagent present certain disadvantages, among them the use of glutaraldehyde for coupling, thereby complicating the composition of the conjugate due to the covalent bonding nature of such a nonselective coupling method. Further, the lectin antibody conjugate is not suitable for the direct passive hemagglutination test and is limited only to solid phase immunoadsorption tests. The nonspecific hemagglutination occasioned by concanavalin A limits the application and sensitivity of this testing procedure.
Another system, utilizing fresh red blood cells as the immunoassay marker or indicator substance, devised for the determination of the presence of hepatitis antigen is described in "Immune Adherence Hemagglutination Test", by M. Mayumi et al., published in Vox Sang, Vol. 20, 1971, p. 178-181. The proposed method utilizes the principle that an antigen-antibody-complement complex has adherence to erythrocytes thus providing a hemagglutination test for hepatitis antigen. The disadvantages of this system are that complement fixation tests in general are not very stable, they are reversible, and they require great technical skill to perform.
In addition to the damage incurred to antibodies by aldehyde treatment as aforementioned, other problems include the rigidity of such fixed cells which limits intercellular contact to a few points thereby producing less force to hold the agglutinate matrix. In contrast, the fresh cells utilized in the present invention have large intercellular contact in numerous areas because of the flexible membrane and consequently exhibit superior holding of the matrix pattern. Thus, higher sensitivity can be reached in the present invention utilizing double antibody conjugate with fresh red blood cells as an indicator substance. A further problem encountered with aldehyde-treated cells is that they become increasingly hydrophobic which in turn causes spontaneous clumping or nonspecific agglutination. These considerations are explored by T. S. Ingraham in Proc. Soc. Exp. Biol. Med., Vol. 99, 1958, p. 452-456.
Although the present invention has great utility as an agglutinating agent of red blood cells, it has the flexibility and capability of using other marker substances in substitution for red blood cells. Such substances include enzymes, radioisotopes, fluorochromes, electron opaque substances and polymeric microspheres which may incorporate fluorescent or nonfluorescent dye materials.
The first immunoglobulin may be selected for its affinity either directly to these marker substances or to another substance linked to the marker substance. The use of enzymes, radioisotopes or electron opaque substances will require more complex procedures and equipment than that necessitated by a red blood cell marker but may nonetheless be desired by the clinician in specialized circumstances.
In addition to those objects already mentioned, it is an object of this invention to provide a diagnostically useful reagent utilizing the double antibody conjugate as direct agglutinating agents useful for the detection of soluble antigens. It is another object of the invention to provide a reagent having maximum antibody avidity permitting the formation of a reagent capable of agglutinating with maximum sensitivity. It is another object of the invention to provide methods and materials which do not require highly specialized handling skills or complex and expensive equipment. It is yet another object of the present invention to provide materials and methods meeting the requirements for third generation sensitivity for hepatitis tests. It is still yet another objective of the present invention to provide a reagent capable of being used with fluorochromes, enzymes, erythrocytes, electron opaque substances, radioactive isotopes, as well as polymeric microspheres. It is another object to provide an agglutinating agent capable of using fresh erythrocytes obtained by the user or supplied in conjunction with the agglutinating agent. It is another object to provide a method for detecting the presence of a specified antigen in a water-soluble or aqueous sample.